Introduction of functional artificial introns into the naturally intronless ura4 gene of Schizosaccharomyces pombe.

Gatermann, K B; Hoffmann, Alexander; Rosenberg, George H.; Käufer, Norbert F.

doi:10.1128/mcb.9.4.1526

Cited by 36 publications

(28 citation statements)

References 54 publications

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“…5 through 7), are as predicted by the intron definition model, and contrast with the patterns of cryptic splice site usage observed in vertebrate pre-mRNAs (re- viewed in reference 6). Our finding that increasing the size of cdc2 intron 2 compromises splicing confirms and extends the results of earlier experiments with an artificial intron in S. pombe, in which expansions also reduced splicing efficiency (24).…”

Section: Discussionsupporting

confidence: 90%

Evidence for Splice Site Pairing via Intron Definition in Schizosaccharomyces pombe

Romfo

Alvarez

Heeckeren

et al. 2000

Molecular and Cellular Biology

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Schizosaccharomyces pombe pre-mRNAs are generally multi-intronic and share certain features with premRNAs from Drosophila melanogaster, in which initial splice site pairing can occur via either exon or intron definition. Here, we present three lines of evidence suggesting that, despite these similarities, fission yeast splicing is most likely restricted to intron definition. First, mutating either or both splice sites flanking an internal exon in the S. pombe cdc2 gene produced almost exclusively intron retention, in contrast to the exon skipping observed in vertebrates. Second, we were unable to induce skipping of the internal microexon in fission yeast cgs2, whereas the default splicing pathway excludes extremely small exons in mammals. Because nearly quantitative removal of the downstream intron in cgs2 could be achieved by expanding the microexon, we propose that its retention is due to steric occlusion. Third, several cryptic 5 junctions in the second intron of fission yeast cdc2 are located within the intron, in contrast to their generally exonic locations in metazoa. The effects of expanding and contracting this intron are as predicted by intron definition; in fact, even highly deviant 5 junctions can compete effectively with the standard 5 splice site if they are closer to the 3 splicing signals. Taken together, our data suggest that pairing of splice sites in S. pombe most likely occurs exclusively across introns in a manner that favors excision of the smallest segment possible.Splice site selection has been most extensively studied in higher eukaryotes (reviewed in reference 11), where abundant evidence indicates that the unit initially recognized by the splicing machinery is the exon, as proposed by Robberson et al. nearly a decade ago (53). Particularly compelling in this regard is the observation that the most common effect of a 5Ј splice site mutation is skipping of the preceding exon rather than inclusion of the mutant intron (61; reviewed in reference 6). Moreover, in the subset of cases in which a 5Ј junction mutation causes activation of a cryptic splice site rather than exon skipping, the new exon-intron boundary is almost invariably located within the preceding exon, again supporting the view that communication occurs across the exon rather than the intron. Finally, there are significant constraints on exon length in vertebrate pre-mRNAs, consistent with the proposal that the 3Ј and 5Ј splice sites on opposite sides of the exon must be recognized concurrently. Not only are the vast majority of natural internal exons in vertebrate pre-mRNAs Ͻ300 nucleotides in length (6), but expanding an exon beyond this size causes it to be skipped (53), particularly if it is surrounded by large introns (60). In contrast to the limitations on exon length, the introns in vertebrate pre-mRNAs can be extremely large (tens of kilobases [29]).Although many questions remain to be answered, several components of the machinery responsible for exon definition have been identified. First, UV cross-linking experiments ...

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Section: Discussionsupporting

confidence: 90%

Evidence for Splice Site Pairing via Intron Definition in Schizosaccharomyces pombe

Romfo

Alvarez

Heeckeren

et al. 2000

Molecular and Cellular Biology

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“…Few S. cerevisiae genes have introns, and in several cases those introns have specific functions: mitochondrial introns encode reverse transcriptases (36) and maturases and endonucleases (13); L32 prevents splicing of its own message by promoting base pairing between the 5' end of the transcript and the 5' splice site (53); and MATal contains two introns that are inefficiently spliced and may play a role in the gene conversion events during mating-type switching (17). S. pombe introns over 180 bp are known to abolish splicing in a model system (19), but the existence of a 700-bp intron in S. pombe p68, which is an essential gene, indicates that this size limit does not operate in vivo. Although it is perhaps not surprising that one of the S. pombe clones was polyadenylated but unspliced, the fact that one of the human cDNA clones was alternately spliced suggests that splicing of the intron is subject to some form of physiological regulation.…”

Section: Discussionmentioning

confidence: 99%

p68 RNA helicase: identification of a nucleolar form and cloning of related genes containing a conserved intron in yeasts.

Iggo¹,

Jamieson²,

MacNeill³

et al. 1991

Mol. Cell. Biol.

124

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The human p68 protein is an RNA-dependent ATPase and RNA helicase which was first identified because of its immunological cross-reaction with a viral RNA helicase, simian virus 40 large T antigen. It belongs to a recently discovered family of proteins (DEAD box proteins) that share extensive regions of amino acid sequence homology, are ubiquitous in living organisms, and are involved in many aspects of RNA metabolism, including splicing, translation, and ribosome assembly. We have shown by immunofluorescent microscopy that mammalian p68, which is excluded from the nucleoli during interphase, translocates to prenucleolar bodies during telophase. We have cloned 55% identical genes from both Schizosaccharomyces pombe and Saccharomyces cerevisiae and shown that they are essential in both yeasts. The human and yeast genes contain a large intron whose position has been precisely conserved. In S. cerevisiae, the intron is unusual both because of its size and because of its location near the 3' end of the gene. We discuss possible functional roles for such an unusual intron in an RNA helicase gene.Manipulation of RNA secondary structure is essential for the proper execution of a large number of processes in the cell. The importance that the cell attaches to this problem has only recently come to light with the discovery of a large family of proteins with putative ATP-dependent RNA helicase activity. These proteins are readily identified because they all possess a core region of highly conserved motifs in their primary amino acid sequences. One of the most conserved motifs is the DEAD box (LDEADxxL), which gives the family its name (34). DEAD box proteins form a subset of a more loosely defined family, identified by Hodgman (27) and Gorbalenya et al. (21), of proteins with known or putative helicase activity.RNA helicase activity has been demonstrated in vitro for only two DEAD box proteins: p68 (26), in which the DEAD motifs were first identified (18), and eIF4A (44), a translation initiation factor. Although the biological processes in which the other members are involved may prove very diverse, it seems reasonable to expect that all of them will show RNA helicase activity, at least in strand displacement assays in vitro. The family appears to be very large, since DEAD box proteins have been found in all prokaryotes and eukaryotes examined, and even the relatively small genome of Saccharomyces cerevisiae contains genes encoding at least 12 different members We are interested in p68 because of its specific immunological cross-reaction with the simian virus 40 large T antigen (31). We have taken two approaches to determine the function of p68. First, we have used immunofluorescent microscopy and antibody microinjection to study the cell biology of p68 in mammalian cells. Using antibodies to * Corresponding author. t Present address:

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“…As expected from this model, mutation of the 5Ј splice site (5Јss) of a short intron leads to intron retention rather than exon skipping, and expansion of short introns inhibits their splicing in vitro and in vivo (4). Short introns inserted into intronless transcripts can be properly spliced, suggesting that the information for splicing of short introns may be contained entirely within the intron (5).…”

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confidence: 99%

A computational analysis of sequence features involved in recognition of short introns

Lim

Burge

2001

Proc. Natl. Acad. Sci. U.S.A.

311

290

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Splicing of short introns by the nuclear pre-mRNA splicing machinery is thought to proceed via an ''intron definition'' mechanism, in which the 5 and 3 splice sites (5 ss, 3 ss, respectively) are initially recognized and paired across the intron. Here, we describe a computational analysis of sequence features involved in recognition of short introns by using available transcript data from five eukaryotes with complete or nearly complete genomic sequences. The information content of five different transcript features was measured by using methods from information theory, and Monte Carlo simulations were used to determine the amount of information required for accurate recognition of short introns in each organism. We conclude: (i) that short introns in Drosophila melanogaster and Caenorhabditis elegans contain essentially all of the information for their recognition by the splicing machinery, and computer programs that simulate splicing specificity can predict the exact boundaries of Ϸ95% of short introns in both organisms; (ii) that in yeast, the 5 ss, branch signal, and 3 ss can accurately identify intron locations but do not precisely determine the location of 3 cleavage in every intron; and (iii) that the 5 ss, branch signal, and 3 ss are not sufficient to accurately identify short introns in plant and human transcripts, but that specific subsets of candidate intronic enhancer motifs can be identified in both human and Arabidopsis that contribute dramatically to the accuracy of splicing simulators. R NA splicing is an essential step in the expression of most eukaryotic genes. An important goal of research on this process is to determine a set of rules that accurately predicts the splicing pattern of primary transcripts. Unlike the process of mRNA translation by the ribosome, which follows a set of rules that is essentially invariant in all known organisms, the rules governing RNA splicing clearly differ between different groups of eukaryotes. Therefore, there is not one but several variants of the ''splicing code'' that remain to be worked out. In addition, the rules for splicing appear to be significantly more complex than those for translation, involving presence of multiple degenerate motifs occurring with appropriate spacing in the transcript. Development of computer algorithms that directly model recognition by the splicing machinery is recognized as an important challenge (1).In human transcripts, the exons are usually short (typically 100-200 bases) and the introns are much longer, averaging about 3 kb (2). The realization that the splicing machinery would face great difficulty in locating splice sites across such long introns led to the exon definition model in which splice sites are paired first across the exons, with spliceosome assembly proceeding through subsequent pairing of exon units (3). The alternative intron definition model derives from the observation that introns in some transcripts (especially in invertebrates) are quite short relative to exons, and so the splicing machinery may initiall...

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Introduction of functional artificial introns into the naturally intronless ura4 gene of Schizosaccharomyces pombe.

Cited by 36 publications

References 54 publications

Evidence for Splice Site Pairing via Intron Definition in Schizosaccharomyces pombe

Evidence for Splice Site Pairing via Intron Definition in Schizosaccharomyces pombe

p68 RNA helicase: identification of a nucleolar form and cloning of related genes containing a conserved intron in yeasts.

A computational analysis of sequence features involved in recognition of short introns

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